CN113652400A

CN113652400A - NK cell serum-free medium and application thereof

Info

Publication number: CN113652400A
Application number: CN202111213232.1A
Authority: CN
Inventors: 林家会; 马士棋; 陈旭; 陈刚
Original assignee: Excell Biology Taicang Co ltd
Current assignee: Suzhou Ecosai Biotechnology Co ltd
Priority date: 2021-10-19
Filing date: 2021-10-19
Publication date: 2021-11-16
Anticipated expiration: 2041-10-19
Also published as: CN113652400B

Abstract

The invention discloses an NK cell serum-free culture medium and application thereof, wherein the NK cell serum-free culture medium is prepared by mixing a basic culture medium and additive components, the additive components comprise rhIL-2, rhIL-15, rhIL-21, tacrolimus, mevalonic acid, isoprene pyrophosphate, recombinant human insulin, recombinant human apotransferrin, recombinant human serum albumin, sodium pyruvate, linoleic acid, cholesterol and folinic acid, and the basic culture medium is IMDM. The culture medium disclosed by the invention is clear in chemical components, free of serum, human-derived and animal-derived components, simple in formula and excellent in performance, and meanwhile, FK506 is added into the serum-free culture medium, so that the high survival rate of cells is ensured, the amplification speed of NK cells can be remarkably increased, the NK cells can be amplified by more than 700 times after being cultured for 17 days, and the cell survival rate can reach more than 95% during harvesting.

Description

NK cell serum-free medium and application thereof

Technical Field

The invention relates to the technical field of culture media, in particular to an NK cell serum-free culture medium and application thereof.

Background

Natural Killer cells, namely NK cells, are lymphocytes which are similar to T cells and B cells but different from the T cells and the B cells, and have a certain regulation effect on the T lymphocytes and the B lymphocytes in human bodies. NK cells are a class of immune cells not associated with a specific immune response, and their mediated lysis is not affected by the Major Histocompatibility Complex (MHC), and are therefore also referred to as the "heart" of the body's natural immune system.

As a first line of defense of the organism, NK cells not only can remove intracellular parasitic bacteria, viruses and aged variant cells, but also have extremely strong removing effect on cancer cells, can realize rapid activation under the condition of no pre-sensitization, and have strong anti-infection and anti-tumor effects. Therefore, the clinical adoptive immunotherapy using NK cells becomes an important means for the tumor cell immunotherapy.

Because the common NK cell culture solution is added with a certain proportion of serum derived from xenogeneic animals, the serum components are complex, and batch-to-batch differences exist, the stability of NK cell culture is easily influenced, and the xenogeneic substances in the serum easily cause xenogeneic animal pathogen infection or residue of xenogeneic animal components, so that certain risks exist in clinical use. Therefore, the use of serum-free medium is the basis for the expansion of NK cells in the clinic. However, most of the existing serum-free culture media are added with components such as human AB serum or human serum albumin, the complexity of the components is increased, the introduction of heterogeneous animal-derived components is avoided, but the serum-free culture media are influenced by the source of raw materials, so that the risk of infecting infectious diseases such as AIDS and hepatitis B can be caused, and the requirements on the quality control of products are greatly improved. And the problems of poor efficiency, poor cell expansion multiple, low biological activity and the like generally exist in the conventional serum-free NK cell culture medium, so that the adoption of the serum-free NK cell culture medium which is safe and reliable and has definite chemical components is very critical to the subsequent clinical application of the serum-free NK cell culture medium.

Patent document CN104928241B, published japanese patent No. 2018.04.13 discloses a culture solution for in vitro activation and amplification of NK cells, which comprises recombinant human insulin, recombinant human transferrin, glutamine, recombinant human albumin, vitamin PP, hydrocortisone, and trace elements. The NK cell culture solution disclosed in the above patent document does not introduce non-human-derived proteins into the whole culture medium system developed, and thus avoids mixing of human-derived components or other animal-derived components into the culture medium. However, in the process of culturing cells, in-vitro human plasma needs to be added, the amplification efficiency is slightly insufficient, and the cell quality needs to be improved. Therefore, there is a need for a serum-free, human-origin, animal-origin component-free NK cell culture medium having high amplification efficiency and having a higher cultured cell quality.

Disclosure of Invention

The invention aims to provide an NK cell serum-free medium and application thereof, so as to solve the problems in the background technology.

In order to solve the technical problems, the invention provides the following technical scheme:

the culture medium comprises a basic culture medium and additional components, wherein the additional components comprise rhIL-2, rhIL-15, rhIL-21, FK506, mevalonic acid, GGPP, recombinant human insulin, recombinant human apo-transferrin, recombinant human serum albumin, sodium pyruvate, linoleic acid, cholesterol and folinic acid.

In an optimized scheme, the contents of the components of the added components are as follows based on the volume of the added components: rhIL-20.1-20 ng/mL, rhIL-1510-100 ng/mL, rhIL-210.1-20 ng/mL, FK 50610-100 ng/mL, mevalonic acid 0.5-5 μ g/mL, GGPP 2-10 μ g/mL, recombinant human insulin 0.01-15 μ g/mL, recombinant apotransferrin 0.1-10 μ g/mL, recombinant human serum albumin 0.1-5 mg/mL, sodium pyruvate 1-100 μ g/mL, linoleic acid 0.05-10 μ g/mL, cholesterol 0.1-10 μ g/mL, and folinic acid 0.05-10 μ g/mL.

In an optimized scheme, the contents of the components of the added components are as follows based on the volume of the added components: rhIL-25 ng/mL, rhIL-1520 ng/mL, rhIL-215 ng/mL, FK 50650 ng/mL, mevalonic acid 1.5 mu g/mL, GGPP 4 mu g/mL, recombinant human insulin 10 mu g/mL, recombinant apotransferrin 5 mu g/mL, recombinant human serum albumin 2mg/mL, sodium pyruvate 50 mu g/mL, linoleic acid 0.2 mu g/mL, cholesterol 1 mu g/mL, folinic acid 0.1 mu g/mL.

In an optimized scheme, the volume ratio of the basic culture medium to the added components is 100: 0.5 to 4.

In an optimized scheme, the volume ratio of the basic culture medium to the added components is 50: 1.

in an optimized scheme, the basic culture medium is an IMDM culture medium.

According to the optimized scheme, the serum-free culture medium is used for culturing the NK cells according to the application of the serum-free culture medium for the NK cells. The serum-free medium is used for culturing NK cells derived from human peripheral blood mononuclear cells.

Compared with the prior art, the invention has the following beneficial effects:

the application makes up the defects of the existing NK cell culture medium, and provides a safe and reliable NK cell chemical component limiting serum-free culture medium containing FK506, mevalonic acid and GGPP, so that the culture purposes of large proliferation quantity, high purity and safe application of NK cells are achieved, and the cultured NK cells can reach the quality safety standard of clinical application.

According to the invention, the function of replacing serum by adding various nutritional factors into the basic culture medium is realized, the chemical components of the culture medium are clear, and the added human insulin, human apotransferrin and human serum albumin are all recombinant expression products, so that the stability among batches is maintained, and no serum is required to be additionally added.

The culture medium disclosed by the invention has the advantages that the addition components with definite components are added into the basic culture medium, so that the components which conflict with each other in the traditional formula are removed, the formula is simplified, the types of the addition components are fewer, the cells are more conveniently cultured, and other cell factors are not required to be additionally added; meanwhile, FK506 is added into a culture solution, high survival rate of NK cells is guaranteed, meanwhile, the NK cells can be rapidly amplified in a large amount, in-vitro human plasma does not need to be added, the obtained NK cells can be subjected to xenogeneic culture, the method is suitable for large-scale preparation and culture of autologous or xenogeneic NK cells, mevalonic acid and GGPP are added, prenylation modification after translation of small G protein in the NK cells is regulated and controlled, immune response of the NK cells can be improved, and accordingly killing performance of the NK cells is remarkably improved.

The NK cell culture medium containing FK506, mevalonic acid and GGPP does not contain human-derived protein components, eliminates the possible pathogenic risk of infectious diseases such as AIDS, hepatitis B and the like in human-derived extracts, uses medicinal-grade components, is safe to human bodies, and is suitable for further scientific research and clinical application research.

The NK cell culture medium containing FK506, mevalonic acid and GGPP has excellent performance, the NK cell can be expanded by more than 700 times in 17 days of culture, the final cell viability can reach more than 95%, the killing rate of the NK cell on K562 cells can reach 50% under the condition of 1:1 effective target ratio in 17 days of culture, the expansion effect is more excellent, and the NK cell culture medium can be widely applied to actual production.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a graphical representation of the viability curves of NK cells in human Peripheral Blood Mononuclear Cells (PBMC) under the chemically defined serum-free medium culture conditions of example 2;

FIG. 2 is a schematic graph of proliferation curves of NK cells in human Peripheral Blood Mononuclear Cells (PBMCs) under the chemically-defined serum-free medium culture conditions of example 2;

FIG. 3 is a graph showing the results of detection of NK cells in human Peripheral Blood Mononuclear Cells (PBMC) by cell markers under the culture conditions of chemically defined serum-free medium of example 2;

FIG. 4 is a schematic diagram showing the detection results of NK cells in human Peripheral Blood Mononuclear Cells (PBMC) for cell markers under commercial serum-free medium culture conditions;

FIG. 5 is a graph showing the proliferation curves of NK cells in human Peripheral Blood Mononuclear Cells (PBMC) under the culture conditions of chemically defined serum-free medium of examples 2 and 4 to 7.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the embodiments of the present application, the basic culture medium is an IMDM culture medium, and the components and the contents of the components of the IMDM culture medium are as follows:

component Chinese name	Component English name	Content (g/L)
			Calcium chloride	Calcium Chloride	0.1653
Magnesium sulfate	Magnesium Sulfate (anhydrous)	0.09767
			Potassium chloride	Potassium Chloride	0.33
Potassium nitrate	Potassium Nitrate	0.000076
			Sodium bicarbonate	Sodium Bicarbonate	3.024
Sodium chloride	Sodium Chloride	4.505
			Sodium hydrogen phosphate	Sodium Phosphate Monobasic (anhydrous)	0.109
Sodium selenite	Sodium Selenite	0.000017
			L-alanine	L-Alanine	0.025
L-arginine hydrochloride	L-Arginine • HCl	0.084
			L-asparagine hydrate	L-Asparagine • H2O	0.0284
L-aspartic acid	L-Aspartic Acid	0.03
			L-cystine hydrochloride	L-Cystine • 2HCl	0.09124
L-glutamic acid	L-Glutamic Acid	0.075
			L-Glutamine	L-Glutamine	0.584
Glycine	Glycine	0.03
			L-histidine hydrochloride hydrate	L-Histidine • HCl • H2O	0.042
L-isoleucine	L-Isoleucine	0.105
			L-leucine	L-Leucine	0.105
L-lysine hydrochloride	L-Lysine • HCl	0.146
			L-methionine	L-Methionine	0.03
L-phenylalanine	L-Phenylalanine	0.066
			L-proline	L-Proline	0.04
L-serine	L-Serine	0.042
			L-threonine	L-Threonine	0.095
L-tryptophan	L-Tryptophan	0.016
			L-tyrosine disodium salt hydrate	L-Tyrosine • 2Na • 2H2O	0.10379
L-valine	L-Valine	0.094
			D-biotin	D-Biotin	0.000013
Choline chloride	Choline Chloride	0.004
			Folic acid	Folic Acid	0.004
Inositol	myo-Inositol	0.0072
			Nicotinamide	Niacinamide	0.004
D-calcium pantothenate	D-Pantothenic Acid (hemicalcium)	0.004
			Pyridoxal hydrochloride	Pyridoxal • HCl	0.004
Riboflavin	Riboflavin	0.0004
			Thiamine hydrochloride	Thiamine • HCl	0.004
Vitamin B12	Vitamin B12	0.000013
			D-glucose	D-Glucose	4.5
N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid	HEPES	5.958
			Pyruvic acid sodium salt	Pyruvic Acid • Na	0.11

The reagents used in the above IMDM medium formulations were purchased from Sigma reagent official website.

Alternatively, the IMDM medium may be prepared by directly purchasing IMDM powder (Sigma, I7633) from Sigma reagent company, adding water for injection and 3.024g of sodium bicarbonate (Sigma, S5761), dissolving sufficiently, and filtering with 0.22 μm filter.

Example 1:

an NK cell serum-free medium comprises a basic medium and an additive component, wherein the basic medium is an IMDM medium, and the volume ratio of the basic medium to the additive component is 1000 mL: 20 mL.

Based on the volume of the added components, the content of each component of the added components is as follows: rhIL-2 (nearshore protein, cat # GMP-CD 66) 20ng/mL, rhIL-15 (nearshore protein, cat # GMP-CD 66) 10ng/mL, rhIL-21 (nearshore protein, cat # GMP-CD 66) 0.1ng/mL, FK506 (MedChemexpress, cat # HY-13756) 10ng/mL, mevalonic acid (Sigma, cat # 90469) 0.5 μ G/mL, GGPP (Sigma, cat # G6025) 10 μ G/mL, recombinant human insulin (Sigma, cat # 91077C) 0.01 μ G/mL, recombinant apotransferrin (Sigma, cat # T1147) 10 μ G/mL, recombinant human serum albumin (protaea, cat # HYC002M 01) 5 mg/mL, sodium pyruvate (Sigma, cat # P5280) 1 μ G/mL, linoleic acid (Sigma, cat # L) 0.3045 μ G/mL, Sigma # 3045 μ G/mL, and gamma 1G/mL, Folinic acid (Sigma, cat No. 47612) 0.05 μ g/mL.

Example 2:

Based on the volume of the added components, the content of each component of the added components is as follows: rhIL-2 (nearshore protein, cat # GMP-CD 66) 5ng/mL, rhIL-15 (nearshore protein, cat # GMP-CD 66) 20ng/mL, rhIL-21 (nearshore protein, cat # GMP-CD 66) 5ng/mL, FK506 (MedChemexpress, cat # HY-13756) 50ng/mL, mevalonic acid (Sigma, cat # 90469) 1.5 μ G/mL, GGPP (Sigma, cat # G6025) 10 μ G/mL, recombinant human insulin (Sigma, cat # 91077C) 10 μ G/mL, recombinant apo transferrin (Sigma, cat # T1147) 5 μ G/mL, recombinant human serum albumin (Heyubo, cat # HYC002M 01) 2mg/mL, sodium pyruvate (Sigma, cat # P5280) 50 μ G/mL, linoleic acid (Sigma, cat # 0.2G/mL), cat # 3045 μ G/mL, Sigma # C3045 μ G/mL, Folinic acid (Sigma, cat No. 47612) 1 μ g/mL.

Example 3:

Based on the volume of the added components, the content of each component of the added components is as follows: rhIL-2 (nearshore protein, cat # GMP-CD 66) 0.1ng/mL, rhIL-15 (nearshore protein, cat # GMP-CD 66) 100 ng/mL, rhIL-21 (nearshore protein, cat # GMP-CD 66) 20ng/mL, FK506 (MedChemexpress, cat # HY-13756) 100 ng/mL, mevalonic acid (Sigma, cat # 90469) 5 μ G/mL, GGPP (Sigma, cat # G6025) 2 μ G/mL, recombinant human insulin (Sigma, cat # 91077C) 15 μ G/mL, recombinant apo-transferrin (Sigma, cat # T1147) 0.1 μ G/mL, recombinant human serum albumin (protaea, cat # HYC002M 01) 0.1mg/mL, sodium pyruvate (Sigma, cat # P5280) 100 μ G/mL, linoleic acid (Sigma, cat # L) 10 μ G/mL, cat # 3045 μ G/mL, Sigma, and gamma G/mL, Folinic acid (Sigma, cat No. 47612) 10 μ g/mL.

Example 4:

an NK cell serum-free medium comprises a basic medium and an additive component, wherein the basic medium is an IMDM medium, and the volume ratio of the basic medium to the additive component is 1000 mL: 10 mL.

Example 5:

an NK cell serum-free medium comprises a basic medium and an additive component, wherein the basic medium is an IMDM medium, and the volume ratio of the basic medium to the additive component is 1000 mL: 30 mL.

Example 6:

an NK cell serum-free medium comprises a basic medium and an additive component, wherein the basic medium is an IMDM medium, and the volume ratio of the basic medium to the additive component is 1000 mL: 40 mL.

Example 7:

an NK cell serum-free medium comprises a basic medium and an additive component, wherein the basic medium is an IMDM medium, and the volume ratio of the basic medium to the additive component is 1000 mL: 50 mL.

Examples 1 to 7 above are any three of the component distribution ratios of the actual culture medium of the present application; the culture medium disclosed in example 2 has a better effect of the ratio of the components, and the culture medium prepared by the culture medium formula disclosed in example 2 is selected for experimental demonstration:

detection test 1: the experimental group of example 2 was used, and a commercial serum-free medium (Irvine Scientific, product number 91215 NK cell medium) was used as a control group;

(1) cell culture plate pretreatment: monoclonal CD16 antibody was diluted to 1 μ g/mL with PBS and T-75cm was added²The flask was incubated at 4 ℃ overnight or in a 37 ℃ incubator for 2 h.

(2) The density centrifugation method is used for freshly separating PBMC from peripheral blood of a healthy person for activating and amplifying NK cells in the invention: adjustment of PBMC Density to 3X 10 Using NK cell chemistry-defined serum-free Medium in the present invention⁶Per mL, the coated T-75cm is taken out²Removing coating solution from the culture flask, adding normal saline along the side wall of the culture flask, gently shaking the culture flask, removing normal saline, adding cell suspension, adding 0.01KE/mL group A streptococcus preparation, standing at 37 deg.C and 5% CO₂Culturing in an incubator.

(3) Observing cell state after 72h, and supplementing fresh NK cell chemical composition limiting serum-free medium when observed cell mass is large and cell culture solution turns yellow, and adjusting cell density to 1.0 × 10⁶/mL, followed by alternate day fluid replacement to maintain the cell concentration at 1.0X 10⁶and/mL, and continuing to culture until day 17 to obtain the cells to be treated.

The detection results are as follows:

detection of cell viability and proliferation efficiency of NK cells

In the culture process disclosed in test 1, samples were taken on

days

0, 3, 5, 7, 10, 12, 14 and 17 of the culture and counted to obtain data on cell proliferation curves and cell viability rates. The cell viability curve is shown in FIG. 1, and the cell proliferation curve is shown in FIG. 2.

And (3) displaying data: on the 17 th day of culture, the NK cells can be expanded by 700 times under the culture condition of the serum-free culture medium (experimental group) prepared in the example 2 of the application, and the cell survival rate reaches more than 95%; is obviously superior to the culture of commercial serum-free culture medium.

2. NK cell surface marker detection

The cellular immunophenotypic assay was performed on day 17 of cell culture during the culture disclosed in test 1. Respectively taking about 1.0 × 10⁶The cells were centrifuged at 2000rpm for 5min, resuspended in PBS after supernatant removal, and incubated with PBS-resuspended cells using fluorescently labeled CD3 antibody (Biolegend, cat # 300439) and CD56 antibody (Biolegend, cat # 362508) under the following conditions: after washing 2 times with PBS at 4 ℃ for 15 min, the supernatant was discarded and resuspended in 0.5 mL PBS. The treated cells were analyzed by flow cytometry.

As shown in FIG. 3 and FIG. 4, wherein FIG. 3 is a schematic diagram of the detection result of cell markers under the culture condition of the culture medium in example 2; FIG. 4 is a schematic diagram showing the detection results of cell markers under commercial serum-free medium culture conditions; and (3) displaying data: NK (CD 3-CD56 +) flow phenotype of up to 82.9% at day 17.

3. NK cell killing activity detection

The K562 cell line which grew vigorously was taken as a target cell, washed 2 times with RPMI-1640 culture medium, and the cell density was adjusted to 1X 10⁶and/mL. 50uL of each well was plated in 96-well plates. NK cells cultured up to day 17 as disclosed in test 1 were collected and adjusted to a cell density of 1X 10⁶/mL、5×10⁶/mL、1×10⁷/mL、2×10⁷and/mL, adding 50uL into a 96-well plate, enabling the effective target ratio to be 1:1, 5:1, 10:1 and 20:1 respectively, and mainly dividing the effective target ratio into four groups, namely an experimental group (K562 + NK), a control group (NK + RPMI-1640), a maximum release group (K562 + RPMI-1640) and a blank group (RPMI-1640), wherein each group comprises 3 multiple wells. And (3) after the inoculated cells are placed in an incubator for co-culture for 24 hours, adding 10 muL of CCK-8 reagent into each hole, shaking up, and continuing to culture for 3 hours in the incubator. And (3) detecting the light absorption value of the enzyme-labeling instrument at the wavelength of 450nm, and calculating the killing rate according to the calculation mode: 1- [ Experimental group (OD value) -control group (OD value) -blank group (OD value) ]/[ maximum release group ](OD value) -blank group (OD value) ]. 100.

As shown in the following table, the NK kill rate at day 17 can reach 100% at the effective target ratio of 10:1 and 20: 1; is obviously superior to the culture of commercial serum-free culture medium.

Effective target ratio	1：1	5：1	10:1	20:1
					EXAMPLE 2 culture Medium	51%	87%	100%	100%
Commercial serum-free culture medium	18%	46%	71%	100%

Detection test 2:

the medium was prepared according to the protocol disclosed in examples 4 to 7, wherein medium No. M1 in example 4, medium No. M2 in example 2, medium No. M3 in example 5, medium No. M4 in example 6, medium No. M5 in example 7, and commercial serum-free medium (Fuji Ohwi science, Inc. Irvine Scientific, Cat. No. 91215 NK cell Medium) were used.

Experiments were performed according to the protocol disclosed in test 1 to differentiate the effects achieved by the media of comparative example 2 and examples 4-7.

The detection results are as follows:

detection of cell viability and proliferation efficiency of NK cells

During the culture process disclosed in the detection test 1, samples are respectively taken and counted on

days

0, 3, 5, 7, 10, 12, 14 and 17 of the culture, and data of cell proliferation curves and cell viability rates are obtained; cell viability data are shown in the following table:

rate of cell viability	D0	D3	D5	D7	D10	D12	D14	D17
									M1	0.91	0.883	0.946	0.817	0.878	0.89	0.919	0.92
M2	0.91	0.92	0.939	0.926	0.942	0.949	0.973	0.958
									M3	0.91	0.899	0.951	0.85	0.855	0.867	0.853	0.87
M4	0.91	0.847	0.897	0.866	0.91	0.85	0.87	0.85
									M5	0.91	0.835	0.899	0.879	0.899	0.909	0.89	0.87
Commercial serum-free culture medium	0.91	0.9	0.862	0.736	0.765	0.777	0.835	0.82

The cell proliferation curve is shown in fig. 5, and the data is shown in the following table:

cell proliferation	D0	D3	D5	D7	D10	D12	D14	D17
									M1	1	1.392857143	5.2	10.15714286	19.05363149	74.64285714	145.43	518.2254317
M2	1	1.678571429	6.421428571	12.61785714	25.06139933	100.4398574	228.22	717.6116305
									M3	1	1.18	4.457142857	9.260714286	16.16113577	92.34934723	180	504.5096675
M4	1	1.028571429	4.278571429	8.028571429	14.33208853	74.64285714	158.32	541.2949139
									M5	1	1.071428571	5.492857143	10.83928571	16.07142857	77.14285714	144.98	485
Commercial serum-free culture medium	1	1.392857143	3.925	6.978571429	13.92857143	30	70	268

And (3) displaying data: on the 17 th day of culture, the expansion amount of NK cells of the M2 preparation scheme is 717 times, the survival rate reaches 95%, and the method is obviously superior to other preparation schemes.

2. NK cell surface marker detection

As shown in the following table, the data show that the M2 formulation NK (CD 3-CD56 +) flow phenotype is 91.2% better at day 17 than the other formulations.

Numbering	CD3 and CD56 expression rates
		M1	74.5%
M2	91.2%
		M3	73.9%
M4	76.9%
		M5	74.8%
Commercial serum-free culture medium	50.6%

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An NK cell serum-free medium, which is characterized in that: the culture medium comprises a basic culture medium and additive components, wherein the additive components comprise FK506, mevalonic acid and GGPP.

2. The NK cell serum-free medium according to claim 1, wherein: the additive components also comprise rhIL-2, rhIL-15, rhIL-21, recombinant human insulin, recombinant human apo-transferrin, recombinant human serum albumin, sodium pyruvate, linoleic acid, cholesterol and folinic acid.

3. The NK cell serum-free medium according to claim 2, wherein: based on the volume of the added components, the content of each component of the added components is as follows: rhIL-20.1-20 ng/mL, rhIL-1510-100 ng/mL, rhIL-210.1-20 ng/mL, FK 50610-100 ng/mL, mevalonic acid 0.5-5 μ g/mL, GGPP 2-10 μ g/mL, recombinant human insulin 0.01-15 μ g/mL, recombinant apotransferrin 0.1-10 μ g/mL, recombinant human serum albumin 0.1-5 mg/mL, sodium pyruvate 1-100 μ g/mL, linoleic acid 0.05-10 μ g/mL, cholesterol 0.1-10 μ g/mL, and folinic acid 0.05-10 μ g/mL.

4. The NK cell serum-free medium according to claim 3, wherein: based on the volume of the added components, the content of each component of the added components is as follows: rhIL-25 ng/mL, rhIL-1520 ng/mL, rhIL-215 ng/mL, FK 50650 ng/mL, mevalonic acid 1.5 mu g/mL, GGPP 4 mu g/mL, recombinant human insulin 10 mu g/mL, recombinant apotransferrin 5 mu g/mL, recombinant human serum albumin 2mg/mL, sodium pyruvate 50 mu g/mL, linoleic acid 0.2 mu g/mL, cholesterol 1 mu g/mL, folinic acid 0.1 mu g/mL.

5. The NK cell serum-free medium according to claim 1, wherein: the volume ratio of the basic culture medium to the additive components is 100: 0.5 to 4.

6. The NK cell serum-free medium according to claim 5, wherein: the volume ratio of the basic culture medium to the additive components is 50: 1.

7. the NK cell serum-free medium according to claim 1, wherein: the basic culture medium is an IMDM culture medium.

8. Use of a NK cell serum-free medium according to any one of claims 1 to 7, wherein: the serum-free medium is used for culturing NK cells.

9. The use of the NK cell serum-free medium according to claim 8, wherein: the serum-free medium is used for culturing NK cells derived from human peripheral blood mononuclear cells.